PACKAGE INSERT For In Vitro Diagnostic Use Only This package insert covers use of: T-SPOT.TB 8 (Multi-use 8-Well Strip Plate Format. Catalogue number: TB.300) T-SPOT.TB 50 (1200 test 96-Well Plate Format. Catalogue number: TB.50) Caution: Federal (USA) law restricts this device to sale by or on the order of a licensed health care professional PI-TB-US-0001 V7 INDEX 1. INTENDED USE ............................................................................................................ 3 2. SUMMARY & EXPLANATION ...................................................................................... 3 Principle of Test ................................................................................................... 4 3. REAGENTS & STORAGE ............................................................................................ 5 Materials Provided ............................................................................................... 5 Storage & Stability ............................................................................................... 5 Equipment and Materials Required but Not Provided ....................................... 6 4. WARNINGS & PRECAUTIONS .................................................................................... 6 5. SPECIMEN COLLECTION & HANDLING .................................................................... 7 6. INSTRUCTIONS FOR USE ........................................................................................... 8 Reagent Preparation ............................................................................................ 9 Cell Counting and Dilution .................................................................................. 9 Plate Set Up and Incubation .............................................................................. 10 Spot Development and Counting ...................................................................... 11 Quality Control ................................................................................................... 12 Results Interpretation and Assay Criteria ........................................................ 12 7. LIMITATIONS ............................................................................................................. 15 8. EXPECTED VALUES .................................................................................................. 16 9. PERFORMANCE CHARACTERISTICS ...................................................................... 17 Assay Performance Characteristics ................................................................. 17 Summary of Clinical Data .................................................................................. 18 Summary of Performance ................................................................................. 26 10. TROUBLESHOOTING .............................................................................................. 34 11. ABBREVIATIONS & GLOSSARY OF SYMBOLS .................................................... 34 12. REFERENCES .......................................................................................................... 36 13. CONTACT INFORMATION ....................................................................................... 37 2 PI-TB-US-0001 V7 1. INTENDED USE The T-SPOT®.TB test is an in vitro diagnostic test for the detection of effector T cells that respond to stimulation by Mycobacterium tuberculosis antigens ESAT-6 and CFP 10 by capturing interferon gamma (IFN-) in the vicinity of T cells in human whole blood collected in sodium citrate or sodium or lithium heparin. It is intended for use as an aid in the diagnosis of M. tuberculosis infection. The T-SPOT.TB test is an indirect test for M. tuberculosis infection (including disease) and is intended for use in conjunction with risk assessment, radiography and other medical and diagnostic evaluations. 2. SUMMARY & EXPLANATION It is estimated that 11 million Americans are infected with M. tuberculosis1. Each person carrying latent TB infection (LTBI) has approximately a 10% chance of progression to active TB disease1. This risk is elevated among certain groups, including those who have been recently infected and those who have clinical conditions that are associated with an increased risk for progression of LTBI to active TB. Historically, TB infection screening was performed with the Tuberculin Skin Test (TST). The recommended technique for administering the TST in the US is the Mantoux method2. The Mantoux skin test involves the intracutaneous injection of tuberculin (also called Purified Protein Derivative – or PPD) into the volar surface of the forearm. TST results are read 48-72 hours after administration; this interval being required for the development of a delayed-type hypersensitivity (DTH) reaction. The extent of induration at the site of injection is measured and recorded in mm. Three different cutoffs for the TST are used to increase its specificity; based on the characteristics of the patient being tested. In addition to the TST, there are ELISA-based assays for detecting IFN- secretion from lymphocytes stimulated with TB-specific antigens3. This method uses a principle similar to that of the T-SPOT.TB test, by measuring the amount of IFN- in blood using the ELISA technique. The T-SPOT.TB test is an in vitro diagnostic test based on an enzyme-linked immunospot (ELISPOT) method. The test enumerates effector T cells responding to stimulation with a combination of peptides simulating ESAT-6 and CFP10 antigens. ESAT-6 and CFP10 are absent from all Bacillus Calmette-Guerin (BCG) strains and from most non-tuberculous mycobacteria with the exception of M. kansasii, M. szulgai and M. marinum4-6. In contrast, individuals infected with M. tuberculosis complex organisms (M. tuberculosis, M. bovis, M. africanum, M. microti, M. canetti) usually have T cells in their blood which recognize these and other mycobacterial antigens. The T-SPOT.TB test can be used in screening of risk groups for TB infection. In addition, the T-SPOT.TB test can be used as a diagnostic aid when evaluating patients suspected of having active TB disease. The T-SPOT.TB test has been tested in some patient groups indicated for screening for TB infection according to current ATS/CDC guidance2: such as, human immunodeficiency virus (HIV) positive persons, recent contacts of TB case patients, residents and employees of high-risk congregate settings, chronic renal failure, children younger than 4 yr of age or infants, children, and adolescents exposed to adults at high-risk and immunosuppressed patients. Refer to the most recent CDC guidance (http://www.cdc.gov/tb/publications/guidelines/testing.htm) for detailed recommendations about diagnosing TB infection (including disease) and selecting persons for testing. 3 PI-TB-US-0001 V7 PRINCIPLE OF TEST The immune response to infection with Mycobacterium tuberculosis is mediated predominantly through T cell activation. As part of this response, T cells are sensitized to M. tuberculosis antigens and the activated effector T cells, both CD4+ and CD8+, produce the cytokine interferon gamma (IFN-) when stimulated by these antigens7-8. The T-SPOT.TB test uses the enzyme-linked immunospot (ELISPOT) methodology to enumerate M. tuberculosis- sensitized T cells by capturing interferon-gamma (IFN-) in the vicinity of T cells from which it was secreted.9 Peripheral blood mononuclear cells (PBMCs) are separated from a whole blood sample, washed and then counted before being added into the assay. Isolated PBMCs (white blood cells) are placed into microtiter wells where they are exposed to a phytohemagglutinin (PHA) control (a mitogenic stimulator indicating cell functionality), nil control, and two separate panels of M. tuberculosis specific antigens. The PBMCs are incubated with the antigens to allow stimulation of any sensitized T cells present. Secreted cytokine is captured by specific antibodies on the surface of the membrane, which forms the base of the well, and the cells and other unwanted materials are removed by washing. A second antibody, conjugated to alkaline phosphatase and directed to a different epitope on the cytokine molecule, is added and binds to the cytokine captured on the membrane surface. Any unbound conjugate is removed by washing. A soluble substrate is added to each well; this is cleaved by bound enzyme to form a (dark blue) spot of insoluble precipitate at the site of the reaction. Evaluating the number of spots obtained provides a measurement of the abundance of M. tuberculosis sensitive effector T cells in the peripheral blood. These principles behind the T-SPOT test system are described in Figure 1 below. Figure 1: Principles of the T-SPOT assay system. For illustration only, refer to Section 6, Instructions for Use for detailed procedural instructions. First a blood sample is collected from the patient. The white blood cells are separated and then put into the wells of a 96-well microtiter plate. The plates are pre-coated with high affinity antibodies ( ) to Interferon-gamma (IFN-γ), a cytokine released by effector T cells when fighting TB infection. Antigens from the TB organism are then added to the wells with the white blood cells to provoke IFN-γ secretion from any effector T cells primed against TB. The antigens are selected to be unique for M. tuberculosis so that only responses of T cells specific for TB are measured. Antigen specific responding T cells ( ) release the cytokine ( ), which is captured in the immediate